Journal: Cell reports

Article Title: Optimizing metastatic-cascade-dependent Rac1 targeting in breast cancer: Guidance using optical window intravital FRET imaging.

doi: 10.1016/j.celrep.2021.109689

Figure Lengend Snippet: Figure 1. Rac1 signaling is increased in MMTV-PyMT-driven metastatic breast cancer (A) Schematic of Rac1-FRET biosensor in FRET conformation upon GTP loading and Rac1 activation in cells crossed to the MMTV-PyMT-driven breast cancer model. (B) Representative images and quantification of upregulated Rac1 activity in primary MMTV-PyMT-driven mammary tumors compared to WT mammary glands (n = 7 mice per condition, 991 cells in total). (C) Spatiotemporal monitoring of live Rac1 activity in the context of the native tumor microenvironment showing the local vasculature (Qdot655) and second harmonic generation (SHG) imaging visualizing the local ECM monitored by optical window imaging in primary tumors (i). Longitudinal imaging can be achieved using these windows and further improved by image stabilization, allowing for the assessment of Rac1 activity in relation to tumor microenvironment (ii). (D) Tracking of Rac1 activity in relation to the proximity of cells to local tumor vasculature, with example excerpts showing cells proximal and distal to the local vasculature (n = 5 mice, 166 cells). (E) Upregulation of Rac1 activity at the invasive border of primary tumors as quantified through an optical window (n = 5 mice, 180 cells). Columns show averages, and error bars represent SEM; scale bars, 50 mm. Unpaired Welch’s t test, ****p < 0.0001 and *p < 0.05 (B and E); one-way ANOVA, *p < 0.05 (D).

Article Snippet: Reagent or Resource Source Identifier Antibodies Mouse monoclonal IgG1 anti-Rac1 Cytoskeleton Cat# ARC03, RRID:AB_10709099 Rabbit poly-clonal IgG anti-GFP Abcam Cat# ab290, RRID:AB_303395 Rabbit monoclonal IgG1 anti-GAPDH Cell Signaling Cat# 2118, RRID:AB_561053 Sheep-anti-Mouse IgG HRP-linked GE healthcare Cat# NA931, RRID:AB_772210 Donkey-anti-Rabbit IgG HRP-linked GE healthcare Cat# NA934, RRID:AB_772206 Rabbit monoclonal IgG1 anti-Ki67 ThermoFisher Scientific Cat# RM-9106-S1, RRID:AB_149792 Rabbit polyclonal anti-cleaved Caspase-3 Cell Signaling Cat# 9661, RRID:AB_2341188 Rabbit monoclonal anti-Cyclin B1 (EPR17060) Abcam Cat# ab181593, RRID:AB_2820245 Rabbit polyclonal anti- Cyclin A2 (C-19) Santa Cruz Biotechnology Cat# sc-596 Rabbit polyclonal anti-PAK1 Cell Signaling Cat# 2602, RRID:AB_330222 Rabbit polyclonal anti-PAK1 (Ser199/204)/PAK2 (Ser192/197) Cell Signaling Cat# 2605, RRID:AB_2160222 Mouse Monoclonal anti-beta-actin (clone AC-15) Merck (SigmaAldrich) Cat# A5441, RRID:AB_476744 Bacterial and virus strains E. coli DH5a ThermoFisher Scientific Cat# 18265017 Chemicals, peptides and recombinant proteins Qtracker 655 Vascular Label ThermoFisher Scientific Cat# Q21021MP AnnexinV-Cy5 BioVision Cat# 1013 Propidium iodide Merck, Sigma-Aldrich Cat# P4170 SalI Promega Cat# R6051 NdeI New England Biolabs Cat# R0111S Rac1 Pull-down Activation Assay Biochem Kit CytoSkeleton Inc. Cat# BK035 Experimental models: Cell lines PyMT 20065 (from mouse 20065, female) SEARCHBreast https://searchbreast.org/ MMTV-PyMT;Rac1-FRET (from mouse 8625, female) This study N/A MMTV-PyMT;Rac1-FRET;P-Rex1 ± (from mouse 138509, female) This study N/A Experimental models: Organisms/strains Rac1-FRET mice (ubiquitously expressed, ‘‘ON’’) Prof. Heidi CE Welch, Babraham Institute, UK, Johnsson et al. 2014 N/A MMTV-Her2 mice The Jackson Laboratory Stock# 002376 MMTV-PyMT mice The Jackson Laboratory Stock# 002374 K14-CreER mice The Jackson Laboratory Stock# 005107 LSL-K-Ras G12D mice The Jackson Laboratory Stock# 008179 Rac1-flox mice Prof L. Machesky, Prof O. Sansom, CRUK Beatson Institute, University of Glasgow N/A P-Rex1 KO mice Prof. Heidi CE Welch, Babraham Institute, UK, Welch et al. 2005 N/A FVB/NJ (Ausb) mice The Jackson Laboratory Stock# 001800 NOD.Cg-Prkdc < scid > IL2rg < tm1Wjl > /SzJ (Ausb) mice The Jackson Laboratory Stock# 005557 BALB/c-Fox1nu (Ausb) mice Charles River Strain# 194 (Continued on next page) Cell Reports 36, 109689, September 14, 2021 e1

Techniques: Activation Assay, Activity Assay, Imaging

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Low Density Lipoprotein Receptor-Related Protein-1 (LRP1) Is Involved in the Uptake of Clostridioides difficile Toxin A and Serves as an Internalizing Receptor

doi: 10.3389/fcimb.2020.565465

Figure Lengend Snippet: Delayed cellular uptake of TcdA upon genetic deletion of LRP1. (A) LRP1 -/- MEFs and LRP1 +/+ mouse embryonic fibroblasts (MEFs) were treated with 100 pM TcdA for the indicated times. The cellular levels of non-glucosylated Rac/Cdc42, total Rac1, pS144/141-PAK1/2, LRP1, and beta actin were analyzed by immunoblotting using the indicated antibodies. (B) Quantification of the relative level of non-glucosylated Rac/Cdc42 versus total Rac1 of time-dependent 100 pM TcdA treated MEFs (C) and time-dependent 3,7 pM TcdB treated MEFs are expressed as the mean ± SD from three independent experiments. * indicates significant differences, p < 0,05 as analyzed using student´s t-test.

Article Snippet: Proteins from cell lysates were separated using 15% polyacrylamide gels und transferred onto nitrocellulose for 2 h at 120 V, followed by blocking with 5% ( w/v ) nonfat dried milk for 1 h. Primary antibodies were incubated over night at 4°C with dilution according to the manufacturers´ instructions ((beta-actin, Mab AC-40, Sigma-Aldrich, St. Louis, MO, USA; dilution 1:5,000); PAK2 (Cell signaling 2608, dilution 1:1,000); phospho-S144/141-PAK1/2 (Abcam ab40795; dilution 1:1,000); Rac1 (BD Transduction Laboratories 610650, clone 102; dilution 1:1,000); Rac1 (Millipore 05-389, clone 23A8; dilution 1:1,000); LRP1 (Abcam ab92544; dilution 1:50,000); TfR (Invitrogen 13-6,800, clone H68.4; dilution 1:1,000); gp96 (R&D Systems 816803, dilution 1:1,000) in TBST buffer (50 mM Tris-HCL, pH 7.2, 150 mM NaCl, 5 mM KCl, 0.05% ( w/v ) Tween 20) and subsequently for 1 h at room temperature with HRP-conjugated secondary antibody (mouse: Rockland 610-1034-121; dilution 1:5,000; rabbit Rockland 611-1302; dilution 1:5,000).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: K-Ras Mediated Murine Epidermal Tumorigenesis Is Dependent upon and Associated with Elevated Rac1 Activity

doi: 10.1371/journal.pone.0017143

Figure Lengend Snippet: ( A ) Macroscopic view of oral papillomas from LSL-K-Ras G12D ;K14-Cre:ER (upper panel) and LSL-K-Ras G12D ;K14-Cre:ER;Rac1 Wt/- mice (lower panel). Deficiency for one Rac1 allele dramatically reduced papilloma growth. ( B ) Kaplan-Meier survival curve of Rac Wt/- (n = 20), LSL-K-Ras G12D ;K14-Cre:ER (n = 78) and LSL-K-Ras G12D ;K14-Cre:ER;Rac1 Wt/- (n = 22) mice shows the dramatic difference between the two mouse strains. Median survival for LSL-K-Ras G12D ;K14-Cre:ER;Rac1 Wt/- mice was determined to be 75 days, which is 50% greater than the 51 day survival time of LSL-K-Ras G12D ;K14-Cre:ER mice. ( C ) Staining for the marker of cell proliferation Ki-67 revealed markedly lower levels in tumors from LSL-K-Ras G12D ;K14-Cre:ER;Rac1 Wt/- mice relative to tumors from LSL-K-Ras G12D ;K14-Cre:ER + mice.

Article Snippet: Antibodies used were against active Rac1-GTP (Neweast Biosciences cat. no.: 26903), Rac1 (Santa Cruz Biotechnology Inc. cat. no.: sc-217).

Techniques: Staining, Marker

Journal: PLoS ONE

Article Title: K-Ras Mediated Murine Epidermal Tumorigenesis Is Dependent upon and Associated with Elevated Rac1 Activity

doi: 10.1371/journal.pone.0017143

Figure Lengend Snippet: ( A ) Active Rac1 was immunoprecipitated from lysates derived from NIH 3T3 cells that had been treated for the indicated time periods with PDGF (10 ng/ml). Western analysis for Rac1 was carried out on immunoprecipitated protein as well as the original lysate as indicated. ( B ) Histogram showing active Rac1 immunofluorescence intensity averaged over 5 fields of NIH 3T3 cells increasing over the indicated time periods of PDGF treatment. ( C ) Staining for actin with Texas-red conjugated phalloidin (red) and co-immunofluorescence for active Rac1 (green) in representative fields of NIH 3T3 cells used in the quantification shown in panel B, demonstrating increased active Rac1 and changes to the actin cytoskeleton over the indicated time periods of PDGF treatment. Boxed areas indicated by numbers 1-3 are shown enlarged. ( D ) Immunohistochemical analysis for Rac1 and active Rac1 of oral mucosa from LSL-K-Ras G12D , LSL-K-Ras G12D ; K14-Cre:ER and Rac1 Wt/- ; LSL-K-Ras G12D ; K14-Cre:ER showing dramatic increase in active Rac1 in LSL-K-Ras G12D ; K14-Cre:ER tissue, which is reduced when one Rac1 allele is lacking. Histogram shows results of positive pixel analysis for active Rac1staining.

Article Snippet: Antibodies used were against active Rac1-GTP (Neweast Biosciences cat. no.: 26903), Rac1 (Santa Cruz Biotechnology Inc. cat. no.: sc-217).

Techniques: Immunoprecipitation, Derivative Assay, Western Blot, Immunofluorescence, Staining, Immunohistochemical staining

Journal: Scientific Reports

Article Title: Rac1 inhibition regenerates wounds in mouse fetuses via altered actin dynamics

doi: 10.1038/s41598-024-78395-2

Figure Lengend Snippet: Expression of Rac1 signaling in the wound during embryonic mouse development. ( a ) Immunostaining of Rac1 in embryonic day (E)13, E15, and E17 mouse fetuses 24 h after wounding. Rac1 is weakly expressed at E13, but is highly expressed in the wound epidermis after E15. Arrows: area of wound. Red: Rac1; blue: DAPI (nuclei). Scale bar = 100 μm. ( b ) Quantitative evaluation of Rac1 gene expression in E13, E15, and E17 mouse fetuses 24 h after wounding. ( c ) Immunostaining of ARP2/3 in E13, E15, and E17 mouse fetuses 24 h after wounding. Weak ARP2/3 expression is visible on E13, whereas high expression is visible in the wound epidermis after E15. Arrows: area of wound. Red: ARP2/3; blue: DAPI (nuclei). Scale bar = 100 μm. ( d ) Quantitative evaluation of ARP2 and ARP3 gene expression in E13, E15, and E17 mouse fetuses 24 h after wounding. * P < 0.05.

Article Snippet: Samples were then incubated with anti-Rac1 antibody (1:100, GTX100761, GeneTex, Irvine, CA, USA), anti-ARP2/3 antibody (1:100, MABT95, Sigma-Aldrich), and anti-desmin antibody (1:1000, MA122150, Thermo Fisher Scientific) diluted with PBS at 4 °C overnight.

Techniques: Expressing, Immunostaining, Gene Expression

Journal: Scientific Reports

Article Title: Rac1 inhibition regenerates wounds in mouse fetuses via altered actin dynamics

doi: 10.1038/s41598-024-78395-2

Figure Lengend Snippet: Effect of NSC23766 treatment on mouse epidermal keratinocyte PAM212. ( a ) Actin dynamics at the PAM212 cell margin in a scratch assay (immunocytochemistry). Scale bar = 10 μm. Red: E-cadherin; green: actin; blue: DAPI (nuclei). Yellow arrowheads: filopodia. ( b ) Expression of Rac1 signaling-related molecules in response to NSC23766 treatment. * P < 0.05. ( c ) Changes in Rac1 activity in response to NSC23766 treatment (Rac1 activation assay). * P < 0.05. ( d ) Quantitative comparison of Rac1 signaling gene expression after NSC23766 treatment. * P < 0.05. ( e ) Effect of NSC23766 treatment on cell migration capacity in the scratch assay. The area covered by migrating cells was measured as the area of migration relative to the area of the gap due to scratching. * P < 0.05.

Article Snippet: Samples were then incubated with anti-Rac1 antibody (1:100, GTX100761, GeneTex, Irvine, CA, USA), anti-ARP2/3 antibody (1:100, MABT95, Sigma-Aldrich), and anti-desmin antibody (1:1000, MA122150, Thermo Fisher Scientific) diluted with PBS at 4 °C overnight.

Techniques: Wound Healing Assay, Immunocytochemistry, Expressing, Activity Assay, Activation Assay, Comparison, Gene Expression, Migration

Journal: Scientific Reports

Article Title: Rac1 inhibition regenerates wounds in mouse fetuses via altered actin dynamics

doi: 10.1038/s41598-024-78395-2

Figure Lengend Snippet: Effect of NSC23766 on wound healing in mouse fetuses. ( a ) Macroscopic analysis of the scar 72 h after the administration of NSC23766 to embryonic day (E)14. The upper panel shows the analysis of 2D photographs and the lower panel shows the analysis of 3D images. Yellow dotted line: area of visible mark. Scale bar = 1 mm. ( b ) Quantitative comparison of wound depth and area 72 h after injury following NSC23766 administration at E14. * P < 0.05. ( c ) Hematoxylin & eosin-stained images of the scar 72 h after the administration of NSC23766 to embryonic day (E)14. Skin structures completely regenerated in the NSC23766-treated group. Black arrows: area of the wound; yellow triangles: tips of the panniculus carnosus muscle. Scale bar = 200 μm. ( d ) Regeneration of panniculus carnosus muscle 72 h after the administration of NSC23766 to embryonic day (E)14 (fluorescent staining of desmin). Red: desmin; blue: DAPI (nuclei); white arrows: area of the wound; yellow triangles: tips of the panniculus carnosus muscle. Scale bar = 200 μm. ( e ) Immunostaining for actin in the wound epidermis at E14 and 36 h after NSC23766 administration. Green: actin; blue: DAPI (nuclei); yellow dotted line: wound margins. Scale bar = 20 μm. ( f ) Evaluation of Rac1 activity in the wound epidermis at E14 at 36 h after NSC23766 administration and wounding. * P < 0.05.

Article Snippet: Samples were then incubated with anti-Rac1 antibody (1:100, GTX100761, GeneTex, Irvine, CA, USA), anti-ARP2/3 antibody (1:100, MABT95, Sigma-Aldrich), and anti-desmin antibody (1:1000, MA122150, Thermo Fisher Scientific) diluted with PBS at 4 °C overnight.

Techniques: Comparison, Staining, Immunostaining, Activity Assay

Journal: Scientific Reports

Article Title: Rac1 inhibition regenerates wounds in mouse fetuses via altered actin dynamics

doi: 10.1038/s41598-024-78395-2

Figure Lengend Snippet: Wound healing in adult epidermis-specific Rac1 -knockout mice (K14-CreERT2;Rac1flox/flox). ( a ) Shema of K14-CreERT2;Rac1flox/flox mouse crosses. ( b ) Evaluation of Rac1 expression in the epidermis. TM: Tamoxifen. * P < 0.05. ( c ) Progression of wound healing in epidermis-specific Rac1 -knockout mice. Tamoxifen treatment significantly prolongs epithelialization in epidermis-specific Rac1 -suppressed mice. Scale bar = 1 cm. * P < 0.05. ( d ) Hematoxylin & eosin stained images of wound healing process in epidermis-specific Rac1 -knockout mice. Scale bar = 100 μm. ( e ) Masson’s trichrome stained image of the wound healing process in epidermis-specific Rac1- knockout mice. Scale bar = 100 μm.

Article Snippet: Samples were then incubated with anti-Rac1 antibody (1:100, GTX100761, GeneTex, Irvine, CA, USA), anti-ARP2/3 antibody (1:100, MABT95, Sigma-Aldrich), and anti-desmin antibody (1:1000, MA122150, Thermo Fisher Scientific) diluted with PBS at 4 °C overnight.

Techniques: Knock-Out, Expressing, Staining

Journal: Scientific Reports

Article Title: Rac1 inhibition regenerates wounds in mouse fetuses via altered actin dynamics

doi: 10.1038/s41598-024-78395-2

Figure Lengend Snippet: Wound healing in fetuses of epidermis-specific Rac1 -knockout mice (K14-CreERT2;Rac1flox/flox). ( a ) Macroscopic analysis of scars in embryonic day (E)14 fetuses 72 h after wounding. The wound was completely regenerated in the tamoxifen group. Yellow dotted line: extent of visible mark. Scale bar = 1 mm. ( b ) Quantitative comparison of wound depth and area 72 h after wounding at E14. * P < 0.05. ( c ) Immunostaining for actin in the wound epidermis at E14 and 36 h after wounding. Green: actin; blue: DAPI (nuclei); arrows: areas where actin is expressed in a cable-like structure. Scale bar = 20 μm. ( d ) Protein expression of Rac1 -signaling-related molecules in E14 embryos from epidermis-specific Rac1 -knockout mice. * P < 0.05. ( e ) Changes in Rac1 activity in fetal E14 epidermis-specific Rac1 -knockout mice (Rac1 activation assay). * P < 0.05.

Article Snippet: Samples were then incubated with anti-Rac1 antibody (1:100, GTX100761, GeneTex, Irvine, CA, USA), anti-ARP2/3 antibody (1:100, MABT95, Sigma-Aldrich), and anti-desmin antibody (1:1000, MA122150, Thermo Fisher Scientific) diluted with PBS at 4 °C overnight.

Techniques: Knock-Out, Comparison, Immunostaining, Expressing, Activity Assay, Activation Assay

Journal: American Journal of Human Genetics

Article Title: Opposite Modulation of RAC1 by Mutations in TRIO Is Associated with Distinct, Domain-Specific Neurodevelopmental Disorders

doi: 10.1016/j.ajhg.2020.01.018

Figure Lengend Snippet: Mapping of the Mutation Sites on the 3D Structure of the TRIO Spectrin 7 Repeat Domain and GEFD1 (A) Species conservation of the residues in TRIO’s seventh spectrin repeat, which is mutated in neurodevelopmental diseases. Identical residues are labeled in red, and similar residues are in blue. The positions of the residues p.Thr1075, p.Arg1078, and p.Asn1080 are boxed in black and indicated on top of the sequence, which encompasses amino acids 1053 to 1091, corresponding to the second α helix of the spectrin repeat. Represented species are Homo sapiens (h), Mus musculus (m), Rattus norvegicus (r), Xenopus laevis (x), Danio rerio (z), Drosophila melanogaster (d), and Caenorhabditis elegans (ce) . (B) Lateral view of the structural model of the seventh spectrin repeat of TRIO. The spectrin domain was modeled based on the crystal structures of human beta2-spectrin (PDB ID 3EDV ) with our sequence alignment and SWISS-MODEL server. Parts of the structure that were modeled with high and low confidence are in blue and cyan, respectively. Mutations within the spectrin domain seen in affected individuals are indicated in red. (C) Axial view of the cross section of the structure at the site of the mutations. Mutations are indicated in magenta. (D) Sequence alignment of the RAC1-specific DH1 domain of TRIO (and KALIRIN) across evolution. Identical residues are labeled in red and similar residues are labeled in blue. The α helices are depicted schematically on top of the sequence alignment. The positions of the alterations p.Glu1299Lys, p.Arg1428Gln, p.Pro1461Leu, p.Pro1461Thr, and p.His1469Arg are indicated in bold and boxed in red. Each mutation affects a highly conserved residue within helices α-1, α-5, and α-6, which make contact with the target GTPase RAC1. Represented species are Homo sapiens (h), Mus musculus (m), Rattus norvegicus (r), Xenopus laevis (x), Danio rerio (z), Drosophila melanogaster (d), and Caenorhabditis elegans (ce) . (E) Structure of the DH1 domain of TRIO (cyan) in complex with the small GTPase substrate RAC1 (green). Mutations within the DH1 domain seen in affected individuals are indicated in red and can be seen to occur at the protein-substrate interface. The complex was modeled with the crystal structures of DH1 (PDB: 1NTY ) and the complex with substrate (PDB: 1KZ7 ). Figures of the protein structures were generated with PyMol. (F) Most of the GEFD1 mutants are affected in their ability to bind to RAC1 N17 (DN). Immunoblot analysis of a Streptavidin pulldown assay of biotinylated TRIO variants. HEK293T cells were transfected with the indicated biotinylated GFP-TRIO variants and RAC1 N17 . TRIO was pulled down with Streptavidin beads, and the co-precipitating RAC1 N17 was detected with a RAC1 antibody.

Article Snippet: Total cell lysates were analyzed with the relevant anti-RAC1 and anti-GFP antibody (Torrey Pines Biolabs, #TP401).

Techniques: Mutagenesis, Labeling, Sequencing, Residue, Generated, Western Blot, Transfection

Journal: American Journal of Human Genetics

Article Title: Opposite Modulation of RAC1 by Mutations in TRIO Is Associated with Distinct, Domain-Specific Neurodevelopmental Disorders

doi: 10.1016/j.ajhg.2020.01.018

Figure Lengend Snippet: The Spectrin Mutants of TRIO Enhance RAC1 Signaling, Neurite Outgrowth, and Lamellipodia Formation in N1E-115 Cells, Whereas the GEFD1 Mutants Are Mostly Impaired in These Processes (A) Immunoblot analysis of HEK293T cell lysates transfected with the indicated GFP-TRIO variants and detected with an anti-GFP antibody (lower panel). PAK1 phosphorylation amounts are detected with a phospho-Ser144 PAK1 antibody (upper panel) and compared to total PAK1 amounts detected with a PAK1 antibody (middle panel). (B) Quantification of the ratio of phospho-PAK1 amounts over total PAK1 expression. PAK1 phosphorylation is used as a readout for the activation of the RAC1 signaling cascade. Data are presented as the mean ± SEM of at least five independent experiments. (C) Quantification of the neurite outgrowth induced by WT or mutant TRIO. Neurite outgrowth is monitored on the basis of the number of cells harboring an extension of at least twice the length of the soma. Data are presented as n-fold change over WT TRIO, which was arbitrarily set to 1. Data are presented as the mean ± SEM of at least five independent experiments. (D) Quantification of lamellipodia formation induced by WT or mutant TRIO. Data are presented as n-fold change over WT TRIO, which was arbitrarily set to 1. Data are presented as the mean ± SEM of at least five independent experiments. Statistical analysis in (B), (C), and (D) were made by one-way ANOVA followed by Dunnett’s test. Asterisks indicate datasets significantly different from WT ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001). (E) Micrographs of N1E-115 cells transfected with the indicated GFP-TRIO variants (green); rhodamine-phalloidin and Hoechst stained the actin (red) and nuclei (blue), respectively. Representative images for each variant type are presented. White arrowheads point to lamellipodia. Scale bar: 20 μm.

Article Snippet: Total cell lysates were analyzed with the relevant anti-RAC1 and anti-GFP antibody (Torrey Pines Biolabs, #TP401).

Techniques: Western Blot, Transfection, Phospho-proteomics, Expressing, Activation Assay, Mutagenesis, Staining, Variant Assay

Journal:

Article Title: The in vivo roles of STEF/Tiam1, Rac1 and JNK in cortical neuronal migration

doi: 10.1093/emboj/cdg413

Figure Lengend Snippet: Fig. 2. Distribution patterns of Rac1 (A), STEF (B), Tiam1 (C) and activated JNK (D) in the cerebral cortex of E15 embryos visualized by specific antibodies. Scale bar, 200 µm.

Article Snippet: Primary antibodies Primary antibodies used in this study were anti-Rac1 (1/50; Upstate Biotechnology), anti-STEF (1/100; Matsuo et al ., 2002 ), anti-Tiam1 (1/100; SantaCruz C-16), anti-active JNK (1/200; Promega), anti-Hu (1/250; Molecular Probes), anti-MAP2 (1/100; Chemicon), anti-GFP (1/1500, polyclonal; Molecular Probes), anti-GFP (1/10, monoclonal; provided by Dr A.Imura), SMI31 (1/1000; Sternberger Monoclonals, Inc.), anti-MAP1B (1/50; SantaCruz), anti-β-tubulin (1/400; Sigma), anti-α-tubulin (1/100; Sigma) and anti-detyrosinated α-tubulin (1/100; Chemicon) antibodies.

Techniques:

Journal:

Article Title: The in vivo roles of STEF/Tiam1, Rac1 and JNK in cortical neuronal migration

doi: 10.1093/emboj/cdg413

Figure Lengend Snippet: Fig. 3. DN effects of Rac1 and STEF/Tiam1 on the developing cerebral cortex. pEGFP (A, D, G, J and M), pN17-Rac1–IRES–EGFP (B, E, H, K and N) or pPHnTSS-STEF–IRES–EGFP (C, F, I, L and O) was introduced into E14 VZ cells. At P0 (A–C) or P4 (D–O), frozen brain sections were examined for EGFP fluorescence (A–F). White lines in (A)–(F) represent pial and ventricular surfaces. After EGFP signals of P4 samples (D–F) were recorded, sections were subjected to HE staining (G–L). (J–L) Higher magnifications of (G)–(I), around the IZ, respectively. Arrows indicate abnormal accumulation of cells in the IZ of N17-Rac1 or PHnTSS-STEF electroporated brains, respectively. Most of these cells were found to be EGFP positive. Each DN form used in this experiment (N17-Rac1, PHnTSS STEF) contains an epitope tag. By immunostaining, expression of each DN form was confirmed in EGFP-positive cells (data not shown). (M–O) Mice subjected to electroporation were killed at P4 to estimate the extent of migration by recording fluorescence intensities of EGFP in distinct parts of the cerebral cortex; layers II–IV, layers V–VI, IZ and VZ/SVZ. Each score represents mean percentage of relative intensity ± SE. (M) n = 5; (N) n = 7; (O) n = 8. Scale bars: 200 µm in (A)–(I); 50 µm in (J)–(L).

Article Snippet: Primary antibodies Primary antibodies used in this study were anti-Rac1 (1/50; Upstate Biotechnology), anti-STEF (1/100; Matsuo et al ., 2002 ), anti-Tiam1 (1/100; SantaCruz C-16), anti-active JNK (1/200; Promega), anti-Hu (1/250; Molecular Probes), anti-MAP2 (1/100; Chemicon), anti-GFP (1/1500, polyclonal; Molecular Probes), anti-GFP (1/10, monoclonal; provided by Dr A.Imura), SMI31 (1/1000; Sternberger Monoclonals, Inc.), anti-MAP1B (1/50; SantaCruz), anti-β-tubulin (1/400; Sigma), anti-α-tubulin (1/100; Sigma) and anti-detyrosinated α-tubulin (1/100; Chemicon) antibodies.

Techniques: Fluorescence, Staining, Dominant Negative Mutation, Immunostaining, Expressing, Electroporation, Migration

Journal:

Article Title: The in vivo roles of STEF/Tiam1, Rac1 and JNK in cortical neuronal migration

doi: 10.1093/emboj/cdg413

Figure Lengend Snippet: Fig. 4. N17-Rac1 did not affect cell division in VZ cells nor differentiation of their progenies. (A and B) BrdU incorporation in VZ cells in animals electroporated with pEGFP (A) or pN17-Rac1–IRES–EGFP (B). Twenty-four hours after electroporation to E14 embryos, BrdU was intraperitoneally administered to the animals for 1 h and animals were killed immediately afterwards. Frozen sections of brains were immunostained with anti-GFP (green) and anti-BrdU (red) antibodies. Arrows indicate cells co-stained with both antibodies. BrdU incorporation rates (BrdU+ cells/EGFP+ cells) in VZ were 27.0 ± 2.2% in (A) and 25.8 ± 1.6% in (B). Scale bar, 20 µm. (C and D) Expression of an early neuronal marker, Hu. E14 embryos were electroporated with the indicated plasmids and killed at E17. Frozen sections were immunostained with anti-GFP (green) and anti-Hu (red) antibodies. White dotted lines represent the boundary of IZ and SVZ. Scale bar, 20 µm. (E–G) Expression of MAP2. E14 embryos were electroporated and killed at P4. Frozen sections were immunostained with anti-GFP (green) and anti-MAP2 (red) antibodies. (E) shows the region around the CP, and (F) and (G) show the region around the IZ. Arrowheads indicate cells co-expressing the transgene and MAP2. Ectopically located cells differentiated to MAP2-positive neurons, although it could not be determined whether these neurons maintained their original layer specificity. Scale bar, 20 µm. (H and I) Morphology of EGFP-positive cells in IZ of E17 brains electroporated with pEGFP or pN17-Rac1–IRES–EGFP at E14. Cells were stained with anti-EGFP antibody to observe detailed morphology. While control cells [arrowheads in (H)] exhibited a spindle-like morphology with a leading process [arrows in (H)] toward the pial surface, N17-Rac1-expressing cells [arrowheads in (I)] showed a round morphology with minor randomly directed processes [arrows in (I)]. Scale bar, 10 µm.

Article Snippet: Primary antibodies Primary antibodies used in this study were anti-Rac1 (1/50; Upstate Biotechnology), anti-STEF (1/100; Matsuo et al ., 2002 ), anti-Tiam1 (1/100; SantaCruz C-16), anti-active JNK (1/200; Promega), anti-Hu (1/250; Molecular Probes), anti-MAP2 (1/100; Chemicon), anti-GFP (1/1500, polyclonal; Molecular Probes), anti-GFP (1/10, monoclonal; provided by Dr A.Imura), SMI31 (1/1000; Sternberger Monoclonals, Inc.), anti-MAP1B (1/50; SantaCruz), anti-β-tubulin (1/400; Sigma), anti-α-tubulin (1/100; Sigma) and anti-detyrosinated α-tubulin (1/100; Chemicon) antibodies.

Techniques: BrdU Incorporation Assay, Electroporation, Staining, Expressing, Marker

Journal:

Article Title: The in vivo roles of STEF/Tiam1, Rac1 and JNK in cortical neuronal migration

doi: 10.1093/emboj/cdg413

Figure Lengend Snippet: Fig. 5. JNK is activated in migrating neurons in the IZ in a Rac1-dependent manner. (A and B) N17-Rac1 suppresses the activation of JNK in the developing cerebral cortex. E14 brains were electroporated with pEGFP (A) or pN17-Rac1–IRES–EGFP (B) and analyzed at E17. Frozen sections were immunostained with anti-EGFP (green) and anti-activated JNK (red) antibodies. Lower panels are higher magnifications of upper panels. Activated JNK was observed in many control pEGFP-transfected cells [arrows in (A)], but rarely in N17-Rac1 expressing cells in IZ [arrowheads in (B)]. To explain this phenomenon, there exists the possibility that the N17-Rac1-expressing cells are undergoing normal differentiation in an ectopic site (the IZ), rather than N17-Rac1 preventing JNK activation. However, this seemed unlikely because these N17-Rac1-expressing cells were not MAP2-positive at this stage (data not shown). Scale bars: 200 µm in upper panels; 20 µm in lower panels.

Article Snippet: Primary antibodies Primary antibodies used in this study were anti-Rac1 (1/50; Upstate Biotechnology), anti-STEF (1/100; Matsuo et al ., 2002 ), anti-Tiam1 (1/100; SantaCruz C-16), anti-active JNK (1/200; Promega), anti-Hu (1/250; Molecular Probes), anti-MAP2 (1/100; Chemicon), anti-GFP (1/1500, polyclonal; Molecular Probes), anti-GFP (1/10, monoclonal; provided by Dr A.Imura), SMI31 (1/1000; Sternberger Monoclonals, Inc.), anti-MAP1B (1/50; SantaCruz), anti-β-tubulin (1/400; Sigma), anti-α-tubulin (1/100; Sigma) and anti-detyrosinated α-tubulin (1/100; Chemicon) antibodies.

Techniques: Activation Assay, Transfection, Expressing

Journal:

Article Title: The in vivo roles of STEF/Tiam1, Rac1 and JNK in cortical neuronal migration

doi: 10.1093/emboj/cdg413

Figure Lengend Snippet: Fig. 8. The possible Rac1 pathway involved in neuronal migration in vivo (see Discussion). X represents a putative Rac1 GEF(s) other than STEF/Tiam1 involved in neuronal migration.

Article Snippet: Primary antibodies Primary antibodies used in this study were anti-Rac1 (1/50; Upstate Biotechnology), anti-STEF (1/100; Matsuo et al ., 2002 ), anti-Tiam1 (1/100; SantaCruz C-16), anti-active JNK (1/200; Promega), anti-Hu (1/250; Molecular Probes), anti-MAP2 (1/100; Chemicon), anti-GFP (1/1500, polyclonal; Molecular Probes), anti-GFP (1/10, monoclonal; provided by Dr A.Imura), SMI31 (1/1000; Sternberger Monoclonals, Inc.), anti-MAP1B (1/50; SantaCruz), anti-β-tubulin (1/400; Sigma), anti-α-tubulin (1/100; Sigma) and anti-detyrosinated α-tubulin (1/100; Chemicon) antibodies.

Techniques: Migration, In Vivo